The present invention relates to a dispensing assembly for liquid droplets of the order of 30 xcexcl in volume and as low as 10 nl or even smaller. Further, the invention is directed towards providing a method for dispensing such liquids and measurement of their properties.
The present invention relates to an assembly for dispensing and aspirating small volumes of liquid as used extensively for drug development in pharmaceutical, medical diagnostics, biotechnology and indeed small droplets of liquid as used extensively for many techniques in industry. Particular examples of this are High Throughput Screening (HTS), Polymerase Chain Reaction (PCR), combinatorial chemistry, microarraying, and proteomics, although obviously not limited to those. In the area of high throughput screening, PCR, proteomics and combinatorial chemistry, the typical application for such a liquid handling system is in dispensing of small volumes of liquids, for example, 1 ml and smaller and in particular volumes around 1 xcexcl and smaller. The invention is also directed to the aspiration of liquids from sample wells so that the liquids can be transferred between wells. The invention relates also to microarray technology, a recent advance in the field of high throughput screening and genomics. Microarray technology is being used for applications such as DNA and protein arrays: in this technology the arrays are created on glass or polymer slides. The fluid handling system for this technology is directed to dispensing consistent droplets of liquids of submicroliter volume. The present invention is also directed to medical diagnostics, for example, for applications such as single nucleotide polymorphism or others.
Development of instrumentation for dispensing of minute volumes of liquids has been an important area of technological activity for some time. Numerous devices for the controlled dispensing of small volumes of liquids (in the range of 1 xcexcl and smaller) for ink jet printing application have been developed over the past twenty five years. More recently, a wide range of new areas of applications has emerged for devices handling liquids in the low microliter range.
The requirements of a dispensing system vary significantly depending on the application. For example, the main requirement of a dispensing system for ink jet applications is to deliver droplets of a fixed volume with a high repetition rate. The separation between individual nozzles should be as small as possible so that many nozzles can be accommodated on a single printing cartridge. On the other hand in this application the task is simplified by the fact that the mechanical properties of the liquid dispensed namely ink are well-defined and consistent. Also in most cases the device used in the ink jet applications does not need to aspire the liquid through the nozzle for the cartridge refill.
For biomedical applications such as High Throughput Screening (HTS), the requirements imposed on a dispensing system are completely different. The system should be capable of handling a variety of reagents with different mechanical properties e.g. viscosity. Usually these systems should also be capable of aspiring the liquids through the nozzle from a well. On the other hand there is not such a demanding requirement for the high repetition rate of drops as in ink jet applications. Another requirement in the HTS applications is that cross contamination, between different wells served by the same dispensing device, be avoided as much as possible.
The most common method of liquid handling for the HTS applications is based on a positive displacement pump such as described in U.S. Pat. No. 5,744,099 (Chase et al). The pump consists of a syringe with a plunger driven by a motor, usually a stepper or servo-motor. The syringe is usually connected to the nozzle of the liquid handling system by means of flexible polymer tubing. The nozzle is typically attached to an arm of a robotic system which carries it between different wells for aspiring and dispensing the liquids. The syringe is filled with a system liquid such as water. The system liquid continuously extends through the flexible tubing into the nozzle down towards the tip. The liquid reagent which needs to be dispensed, fills up into the nozzle from the tip. In order to avoid mixing of the system liquid and the sample liquid and therefore cross-contamination, an air bubble or bubble of another gas is usually left between them. This method does not allow reliable dispensing of droplets in the volume range below some 1 to 5 microliters. Somewhat smaller volumes can be dispensed if the tip of the dispenser touches the substrate to release the drop. The compressibility of the gas bubble between the reagent and the system liquid is a significant source of error. Examples of such positive displacement pumps are shown in U.S. Pat. No. 5,744,099 (Chase et al). Similarly the problems of dispensing drops of small volume are also described and discussed in U.S. Pat. No. 4,574,850 (Davis) and U.S. Pat. No. 5,035,150 (Tomkins).
Dispensing of drops of liquids using a conventional solenoid valve is well known. It has been used in ink printing applications for more than a decade. As explained below, there are still major problems associated with the use of a conventional solenoid valve for dispensing of minute droplets of reagents for biomedical and pharmaceutical applications.
U.S. Pat. No. 5,741,554 (Tisone) describes another method of dispensing submicroliter volumes of fluids for biomedical application and in particular for depositing bodily fluids and reagents on diagnostic test strips. This method combines a positive displacement pump and a conventional solenoid valve. The positive displacement pump is a syringe pump filled with a fluid to be dispensed. The pump is connected to tubing at the other end which there is a solenoid valve located close to the ejection nozzle. The tubing is also filled with the fluid to be dispensed. In this method the piston of the pump is driven by a motor with a well-defined constant speed. The speed determines the flow rate of the fluid from the nozzle provided the solenoid valve is opened frequently enough and the duty cycle between opening and closing of the valve is long enough. The solenoid valve is actuated with a defined repetition rate. The repetition rate of the valve and the flow rate of the pump determine the size of each drop. For example, if the pump operates at a flow rate of 1 xcexcl per second and the repetition rate is 100 open-close cycles per second, then the size of each drop in theory is 10 nl. This method is suitable for dispensing of large number of identical droplets. However, for dispensing of liquids for HTS applications, this method is often inappropriate since it is commonly required to aspire a liquid through the nozzle in small quantities (say 1 xcexcl) and then dispense it in fractions of this quantity, say in a series of only five drops or even a single drop on demand. To avoid mixing of the liquid aspired with the one in the syringe pump, it is probably necessary to place a bubble of gas in the tube with the attendant problems described above. Without such a bubble, if the solenoid valve open time and/or operating frequency are too small for a given pump flow rate, the pressure in the dispenser will become too great, causing possible rupture or malfunctioning of the system. Another disadvantage of this solution is that the heat from the coil actuating the plunger of the valve may cause a heating of the liquid in the valve that can be a serious problem for some applications. Besides, for some regimes of operation the drops may amalgamate, e.g. one drop will be released for every two or three actuations of the valve.
As the solenoid valve is normally not used as a disposable element due to its high cost, the used portion or potentially contaminated chamber of the valve needs to be washed frequently to avoid cross contamination. This is a major issue for HTS applications and microarraying as the dispenser typically switches from one liquid to another up to several times a minute. The fluid path in the valve is torturous, the valve contains a number of parts and pockets where the contamination can build up complicating the cleaning routine.
Various attempts in the past have been made to address the problem of such conventional solenoid valves. A typical example of these is the invention described in PCT Patent Specification No. WO 99/42752 (Labudde). This patent specification discusses the problems associated with using conventional solenoid valves for many HTS applications. Various solutions to the problem are proposed. None of these, it is suggested, overcome the major problems of the use of conventional solenoid valves for these applications. Relatively complex constructions of actuator and plungers with diaphragms are described. The invention of this patent specification is directed to the problem of bubble formation in the valve and aspiration. WO 99/42752 (Labudde) discusses in some detail the problems relating to the structure and geometry of the valve. The solution proposed is to design a xe2x80x9cnon torturousxe2x80x9d flow path for the liquid. In this patent specification, the effect of the use of a blunt or rounded valve seat is discussed as well as the effect of the area of the valve seat orifice opening. This specification discloses a valve seat with an internal diameter of the order of 7.5 mm. Further, in this latter patent specification, the plunger of the valve is attached to a diaphragm limiting its movement. The displacement of the plunger between the open and closed position is of the order of 50 xcexcm. There is also a discussion in this patent specification of the heating effects and a solution is proposed by separating the actuating coil from the valve. U.S. Pat. No. 5,741,554 (Tisone) again describes substantially the same construction.
Patent Specification No. WO 98/52640 (Shalom) describes a flow control device for medical infusion systems. These systems are used for the slow injection of relatively large volumes, namely milliliters up to a liter, into a patient over a relatively long period of minutes, if not hours. Essentially, there is described a system for the slow injection of fluids into a patient with real time control of the process. The system uses valves to mix or select fluids coming from a number of inlets and to route their flow via selected outlets. It is suggested that this specification does not teach that such a valve would be suitable for the dispensing of droplets of liquid with volumes of the order of 5 nl at a high frequency. In this patent specification, there is illustrated an actuation coil embedded in the body of the valve and the use of spherical magnetic bosses or a multiple of bosses, to increase the resistance of fluid flow through the valve.
U.S. Pat. No. 5,758,666 (Larson et al) describes a surgically implantable reciprocating pump having a floating piston made of a permanent magnetic material and incorporating a check valve. The piston can be moved by means of energising coils in a suitable timing sequence. The piston allows the flow of liquid through it when it moves in one direction as the check valve is open, and when it moves in the opposite direction, the check valve is closed and the liquid is pumped by the piston.
U.S. Pat. No. 4,541,787 (DeLong) describes an electromagnetic reciprocating pump with a xe2x80x9cmagnetically responsivexe2x80x9d piston so called as it contains some ferromagnetic material. The piston is actuated by at least two coils located outside the cylinder containing the piston. The coils are energised by a current with a required timing.
It has become apparent that accurate control of the valve boss is all important. Indeed, there are known linear and rotary motors in which movement of a piston or a shaft of a permanent magnetic material is controlled by a series of driving coils. To achieve reliable operation of the motor, signals applied to coils must be synchronised with the movement of the shaft. For example, there can be ten or so driving coils spaced apart and positioned along the length of the motor to achieve a significant stroke of the shaft. It is clear that at any given moment only those one or two coils that are positioned close to the shaft must be energised and the one or two coils located just behind the shaft must be de-energised. As the shaft moves, successive one or two coils located further down its path are energised and so on. Therefore the motor controller must follow movement of the shaft and must be aware of its current position. This is achieved by detecting electromotive force induced in the driving coils. U.S. Pat. No. 4,965,864 (Roth et al) is an example of this type of motor. This patent however, does not deal with dispensing of small volumes of liquids.
There are also known other devices for the detection of movement of a magnetic shaft. In a typical embodiment disclosed in European Patent Specification No 457,389 A1 (van Alem), there are three coils driving a shaft of a magnetic material. The coils are positioned along the length of the shaft""s path. The central of the three driving coils is also supplied with an AC current. Electromagnetic coupling between the driving coils depends on the position of the shaft. This patent does not deal with dispensing of small volumes of liquids. The device described in this patent specification falls under the category of devices called Linear Variable Differential Transformers (LVDT). These devices are used as position sensors and are manufactured by a number of companies such as e.g. Solartron Metrology Ltd (LVDT type SMI manufactured by Solartron Metrology Ltd, Steyning Way, Southern Cross Industrial Estate, Bognor Regis, PO22 9ST West Sussex, UK).
In summary, there is a major problem in finding a suitable way of dispensing submicroliter volumes for applications as described above such as HTS applications. This problem can be said to be currently the bottleneck in changing to assay formats of higher density. Numerous publications in the specialised literature indicate that a technical solution to this problem has not been found so far and further that it is necessary to find them. Our work to date indicates that if accurate dispensing of submicroliter volumes is to be achieved, it can only be done with solenoid valves having a floating boss.
Other methods of dispensing of small volumes of liquids were proposed recently in various patent specifications (EPO 00650123.3 and EPO 99650106.0, Shvets). The methods are based on a floating boss valve. In a typical dispensing assembly, the boss, made of a ferromagnetic material, is placed inside a body member of a dispenser mounting a nozzle. The boss is actuated by an external magnetic field. As a result the boss can close and open the nozzle bore of the dispenser. The liquid in the dispensing assembly is pressurized by a pressure source. When the boss is removed from the nozzle bore, the liquid is transported by pressure towards the dispensing tip resulting in a dispensing of a drop or droplet of the liquid stored in the dispensing assembly. The source of the magnetic field actuating the boss is located adjacent to the body member of the dispensing assembly and the magnetic boss. It could consist of a magnetic coil or an assembly of magnetic coils or coils wound on a core of magnetic material. Alternatively it could consist of an assembly of permanent magnets coupled by magnetostatic forces with the boss. Such a magnetic assembly can be caused to move by using, for example, a pneumatic actuator thus causing the movement of the boss. The means are also provided to detach the droplet from the nozzle by using the electrostatic field generated at the end of the tip. There are also means provided to measure the mass of the droplet through the measurement of the charge carried by the droplet. There are also means provided for navigating droplets to desired locations within the well plate. It should be pointed that there are also other embodiments described in the patent applications EPO 00650123.3 and EPO 99650106.0 that are not based on a floating boss. The common feature of most embodiments is a soft compressible seal between portion of a capillary forming a nozzle bore and a boss or plunger. This portion of the nozzle forms a valve seat. In the preferred embodiment of EPO 00650123.3, the compressible seal is formed between a valve seat formed by part of a rigid capillary forming the nozzle bore and a soft polymer attached to the surface of the boss/plunger coming in contact with the capillary. In most embodiments there is also a stopper located inside the body member of the dispenser to ensure that the boss always remains positioned in the area where it can be efficiently coupled to the source of magnetic field actuating the boss. It is clear from the technical disclosure of these patent applications that the accuracy of dispensing critically depends on the accuracy of the time interval during which the nozzle bore is open during the dispensing. The time interval during which the nozzle bore is open, is determined by the pattern of the current in the coil assembly actuating the boss/plunger.
Unfortunately it is difficult to predict and thus know the moment of opening and closing of the valve seat even if the current actuating the boss is known. This is caused by the fact that one of the two elements: the boss or the valve seat, must be made of a soft material to ensure that the seal of the boss against the nozzle bore is pneumatically tight. This soft material is compressed when the boss is pressed against the nozzle bore. As a result, movement of the boss away from the valve seat at the start of the dispensation does not immediately result in the opening of the valve seat and flow of the liquid through the nozzle bore. There is a time delay between the start of the movement of the boss and the separation from the valve seat that depends mainly on the compressibility of the seal and the pressure in the dispenser. This time delay between the two events can have a particularly significant effect on the accuracy of dispensing for drops of small volume, as the opening time required to deliver a small drop to the end of the nozzle is particularly short. Indeed, for dispensing of drops in the volume range of 10 nl, the desired opening time interval of the dispenser could be as short as some 1 ms or even smaller. This time depends on the diameter of the nozzle, length of the capillary and viscosity of the liquid dispensed. For droplets of submicroliter volume, the inaccuracy in the control of the moment of opening of the valve seat can, in some circumstances, be a significant source of error.
Additional loss of accuracy of dispensing comes at the moment of closing of the valve seat. The reason is that at the moment when the current in the actuating coil is sent to close the valve seat, the position of the boss is not known. For example if the valve was only open for a short time prior to the moment of closure, the boss would not move far away from the valve seat and would not come to rest against a stopper. Therefore in this case it would take it a shorter time to reach back the valve seat and close the valve than if it was pressed against the stopper. The position of the boss is difficult to predict on the basis of the time that has elapsed since the moment of opening of the valve. The position will depend on a number of factors including, for example, viscosity of the liquid in the dispenser. If the time between the moments of opening and closing of the valve boss is sufficiently long so that the boss has come in contact with the stopper, an additional source of error will come from the fact that the boss can bounce back from the stopper. If the boss bounces back from the stopper, then in contrast to common sense expectations, increasing of the time interval during which the boss is open could, as we have found, result in decrease in the volume of the liquid dispensed. This is discussed in more detail below.
Another source of error comes from the fact that the boss can also bounce back when it reaches the valve seat. If the bouncing is significant, then this could result in an additional uncontrollable opening of the valve and therefore, additional uncontrollable delivery of the liquid through the nozzle to the tip.
Most of these factors become more significant for droplets of small volume for at least two reasons. Firstly, the opening time of the valve for droplets of small volume is shorter than for droplets of large volume and the volume of the drop is proportional to the opening time of the valve. Therefore the time inaccuracies associated with bouncing of the boss and compressibility of the valve seal increase relatively in comparison with the opening time of the valve. Secondly, to ensure a shorter opening time of the valve, the boss must move faster. Therefore, as the velocity of the boss increases, so will the tendency of the boss to bounce back from both the valve seat and the stopper.
It would be advantageous to measure properties of the liquid during the dispensing such as, for example, its viscosity. Simplistically, if one knew the viscosity of the liquid, then one would know firstly when the liquid had become diluted or had changed its properties or alternatively one would know, for example, when there was no liquid in the dispenser. Most of the current dispensing technologies do not allow for this kind of measurement. The present invention is directed towards providing an improved method and apparatus for dispensing droplets as small as 10 nl (10xe2x88x928 l) or even smaller, while at the same time it should be possible to dispense larger droplets such as those as large as 10 micro liters or even greater.
Another objective is to provide a method where the quantity of the fluid dispensed can be freely selected by the operator and accurately controlled by the dispensing system. The system should be capable of dispensing a drop of one size followed by a drop of a widely differing size, for example, a 10 nl drop followed by a 500 nl one.
The invention is also directed towards providing a method where the fluid can be dispensed on demand, i.e. one quantity can be dispensed at a required time as opposed to a series of dispensations with set periodic time intervals between them. Yet, the method should also allow for dispensation of doses with regular intervals between subsequent dispensations, for example, printing with liquids.
Another objective of the present invention is to provide a method and a dispensing device suitable for dispensing a liquid from a supply line to a target well and also for aspiring a liquid from the sample well into the supply line. The device should ideally be able to control accurately the amount of the liquid aspired into the nozzle of the dispenser from a supply well.
Another objective is to provide a method for handling fluids in a robotic system for high throughput screening or microarraying which would be suitable for accurate dispensing and aspiring volumes smaller than the ones obtainable with current positive displacement pumps.
Yet another objective is to provide means of measurement of viscosity and density of the liquid dispensed during the dispensations. Another objective is to enable detection of the moment when the dispenser runs out of liquid and requires refill.
Exemplary embodiments of the present invention provide a dispensing assembly for liquid droplets of the order of 30 xcexcl or less in volume and also various control circuits for such a dispensing assembly as well as a method of dispensing liquid droplets of the order of 30 xcexcl or less in volume in a dispensing assembly using a pressurised delivery source.
One such dispensing assembly comprises a pressurised liquid delivery source and a dispenser having a metering valve body connected to the liquid delivery source, the body being generally an elongate body member having an internal main bore and a base forming an apertured valve seat. The metering valve body has mounted in its bore, a floating valve boss, that is to say, a valve boss that is not connected to any external rod or actuator. The cross-sectional area of the valve boss is sufficiently less than that of the main bore to permit the free passage of liquid therebetween. The dispenser carries a nozzle mounted on the base of the body member which nozzle comprises a dispensing tip and has a nozzle bore communicating between the valve seat and the dispensing tip. A variable power output valve actuator is used for moving the valve boss in the bore between a closed position engaging the valve seat and an open position spaced-apart from the valve boss. Usually, an end stopper will be provided to retain the valve boss within the metering valve body and in many instances will define the open position.
The invention provides a valve boss detector for determining the movement of the valve boss within the bore. This may be a position sensor, it can be a velocity or acceleration sensor, namely, any movement sensor generally. Further, there is provided a controller connected to the valve boss detector and to the valve boss actuator for varying the power input to the valve boss actuator depending on the movement of the valve boss within the bore. Thus, by use of the detector, much more accurate dispensing can be obtained with a floating valve boss, the advantage of the invention being to ensure that there is accurate motion control of the valve boss. It is much more effective than a conventional solenoid valve.
Ideally, the nozzle or at least that portion of it adjacent the tip, is manufactured of a hydrophobic material to encourage separation of the droplet from the dispensing tip. The valve boss will generally be a ferromagnetic material and the actuator will often be a current actuating coil assembly. Various valve boss detectors such as Hall sensors may be provided. The actuator can be one actuating coil, it can be a pair of spaced-apart actuating coils and the sensors may be, for example, two spaced-apart sensing coils. The position sensor, forming part of the valve boss detector, may, for example, be an alternating current powered sensing coil or coils. A parametric oscillator circuit may be used in the position sensor. More than one sensing coil may be used.
In one embodiment of the invention, the dispensing assembly includes a plurality of nozzles mounted on the base of the one body member. Various detection circuits may be used.
Further, the invention provides a method of dispensing liquid droplets of the order of 30 xcexcl or less in volume in a dispensing assembly using a pressurised delivery source feeding a metering valve body, the valve body comprising an elongate body member having an internal main bore, a base forming an apertured valve seat and a boss stopper in the main bore spaced-apart from the base; a valve boss comprising ferromagnetic material in the main bore, the cross-sectional area of which is sufficiently less than that of the main bore to allow the passage of liquid therebetween, a nozzle mounted on the base comprising an elongate needle-like member having a nozzle bore communicating between the valve seat and a dispensing tip and a variable power actuator for moving the valve boss in the bore between a closed position in engagement with the valve seat and an open position spaced-apart from the valve seat, the variable power actuator moving the valve boss by applying an electrical field to the valve boss, recording the position of the valve boss within the main bore of the body member throughout the dispensing cycle and using the information on the position of the valve boss within the bore to vary the force to be exerted on the valve boss between a highest force on engagement and disengagement of the valve boss with the valve seat and a lower force needed to keep the valve boss in spaced relationship with the valve seat to maintain the valve open. Needless to say, the lowest force will be zero at some instance when changing direction of the boss.
Then by opening the metering valve by exerting a disengaging force to separate the valve boss from the valve seat, once it has been separated, that is to say, on sensing of the opening of the metering valve, a lower opening force is exerted to remove the valve boss to a fully open position remote from the valve seat. Generally, this will be against, but does not have to be against, a valve stopper. It will largely depend on the volume of the droplet being dispensed and on the general configuration of the valve body. Then a lower force will be maintained to keep the valve open to retain the valve boss in a fully open position. Then, when it is required to close the valve, a closing force will be exerted on the valve boss to move the valve boss close to the valve seat. Then when the valve boss is sensed as being about to contact the valve seat, then a valve engaging force is exerted on the valve boss and this force must be such as to ensure that the valve boss does not bounce on the valve seat and then, on sensing that the valve boss is stationary, a lower closed maintaining or retaining force is exerted on the valve boss. Thus, overheating is largely prevented. Obviously, when there is a boss stopper, then additionally, when the valve boss is sensed as about to contact the valve stopper, there is exerted a variable stopper engaging force in the valve seat to cause the valve boss to attain zero boss velocity or essentially to stop. Then, again, once the valve boss is contacting a valve stopper, once it has stopped and is stationary, then a lower force is exerted on the valve boss.
Further, the invention provides methods of determining various characteristics of the liquid being dispensed. For example, it is possible to measure the volume of the droplet being dispensed by measuring the difference between the opening force and the closing force. The volume may be calculated from various formulae such as:
Vdisp=U1*Sg*t
and
Fmoxe2x88x92Fmc=k*xcex7*2*U1,
xcex7=viscosity of fluid.
k=constant dependent on boss and main bore dimensions.
U1=flow velocity in the gap between the boss and the body member due to the delivery of liquid to the tip of the dispenser.
Sg=cross sectional area of gap between the valve boss and the main bore.
Fmo=opening magnetic force.
Fmc=closing magnetic force.
t=time during which the dispenser is open.
Alternatively, the volume may be calculated from:
Vdisp=Qnozzle*t
where
Qnozzle is flow rate through nozzle bore
t=time during which the dispenser is open
and Qnozzle=xcfx80*xcex4P*rn4/(8*Ln*xcex7)
where
xcex4P=pressure difference along nozzle from seat to tip
rn=nozzle bore radius
Ln=nozzle length
xcfx80=3.1415.
Further, the invention provides a method of measuring the density of a liquid being dispensed by measuring the current required to move the boss at a constant relatively low velocity and the density is calculated from
l*km=Vb*g*(Pbxe2x88x92Pl)
l=current in the actuating coil assembly
km=coefficient dependent on actuating coils
Vb=volume of boss
Pb=density of boss
Pl=density of liquid
g=acceleration due to gravity